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Comparative Study
. 2007 Jun;14(6):475-83.
doi: 10.1038/nsmb1251.

Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats

Owen Richard Davies  1 , Luca Pellegrini
Affiliations
Comparative Study

Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats

Owen Richard Davies et al. Nat Struct Mol Biol. 2007 Jun.

Erratum in

  • Nat Struct Mol Biol. 2007 Jul;14(7):680

Abstract

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes [corrected] of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.

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Figures

Figure 1
Figure 1
Structure and function of BRCA2 and RAD51 proteins. (a) Schematic diagram of the human BRCA2 protein. The RAD51-binding regions in the middle and C-terminus of the protein, as well as its DNA-binding domain, are indicated. (b) Recombinases of the RecA/RAD51/RadA family oligomerise to form rings or filaments. Representative examples of both arrangements are shown, as observed in the crystal structures of Pyrococcus furiosus RadA (PDB entry 1PZN) and Methanococcus voltae RadA (pdb entry 2F1H), respectively (adjacent subunits are shown in orange and blue). In the oligomeric forms, the recombinase protomers associate through an evolutionarily-conserved mechanism. (c) BRC repeat binding can disrupt RAD51 oligomers and nucleoprotein filaments, by sequestering RAD51 in a monomeric form. (d) Multiple sequence alignment of the RAD51-binding sequence coded by exon 27 of vertebrate BRCA2 (absolutely conserved residues are shown in red and partially conserved residues in yellow; numbering refers to the human BRCA2 sequence). The RAD51-binding sequence overlaps with a cyclin-binding motif that enables CDK-dependent phosphorylation of residue Ser3291 at the G2-M phase transition of the cell cycle. The extent of the BRCA2Exon 27 sequence used in this work is indicated. Please note that the schematic diagrams shown in this and subsequent figures are meant to illustrate the overall effect of BRCA2 peptides upon RAD51 oligomerisation. However, they are not intended to represent the actual number of protomers present in oligomeric RAD51 or the stoichiometry of the BRCA2Exon 27–RAD51 interaction.
Figure 2
Figure 2
BRCA2Exon 27 binds to oligomeric RAD51. (a) Streptavidin pull-down of RAD51 with biotinylated BRCA2Exon 27, BRCA2Exon 27-Ph and BRC4 peptides, visualized by anti-RAD51 western blot. RAD51 binds to BRC4 and BRCA2Exon 27 but not to the BRCA2Exon 27 phosphorylated on Ser3291. (b) Native gel electrophoresis of RAD51 upon addition of increasing amounts of BRC4. (c) Native gel electrophoresis of RAD51 upon addition of increasing amounts of BRCA2Exon 27. (d) Native gel electrophoresis of fluorescein-labeled Fl-BRCA2Exon27 upon addition of increasing amounts of RAD51. A molar excess of RAD51 (lane 7) induces a complete shift of Fl-BRCA2Exon 27 to the position of oligomeric RAD51. The intermediate mobility of Fl-BRCA2Exon 27 in the presence of lower amounts of RAD51 (lanes 5-6) likely corresponds to RAD51–BRCA2Exon 27 complexes that have wholly or partially dissociated during electrophoresis.
Figure 3
Figure 3
Cross-linking of RAD51, RAD51–BRC4 and RAD51–BRCA2Exon 27 complexes. (a) Complexes between RAD51 and BRC4 or BRCA2Exon 27 were cross-linked by BS3 and the resulting species were analyzed by SDS-PAGE. (b) The native oligomeric state of RAD51 is evident through the formation of several multimeric species. (c) An excess of BRC4 disassembles the RAD51 oligomer through the formation of a 1:1 complex between one BRC4 peptide and a RAD51 protomer. (d) SDS-PAGE of the cross-linked complex between RAD51 and Fl-BRCA2Exon 27 visualized by Coomassie staining. (e) SDS-PAGE of the cross-linked complex between RAD51 and Fl-BRCA2Exon 27, exposed to UV radiation to visualize the fluorescein-labeled peptide.
Figure 4
Figure 4
BRCA2Exon 27 binding requires an oligomeric RAD51 assembly. (a) The conserved oligomerisation interaction between the linker region of one protomer and the ATPase domain of the adjacent protomer is visualized in this view of two adjacent subunits of the Pyrococcus furiosus RadA ring structure (PDB entry 1PZN). Mutation of the interface residue Phe86, equivalent to Phe96 of P. furiosus RadA, provides a constitutively monomeric form of human RAD51. Furthermore, a construct of RAD51 containing residues 80-339 (Lys80 RAD51) lacks the N-terminal domain but retains the integrity of the oligomerisation interface. Streptavidin pull-downs of (b) F86E RAD51, (c) GST–F86E RAD51 fusion protein, and (d) GST–RAD51 fusion protein with biotinylated BRCA2Exon 27, BRCA2Exon 27-Ph and BRC4 peptides, visualized by anti-RAD51 western blot. (e) Gel filtration analysis of Lys80 RAD51, and Lys80 RAD51 incubated with excess BRC4, visualized by an anti-RAD51 dot-blot of eluted fractions. A control gel filtration run was performed using the previously described BRC4–S97 RAD51 fusion protein, which approximates a monomeric complex between BRC4 and Lys80 RAD51. The elution positions of gel filtration standards are shown adjacent to the dot-blot. (f) Streptavidin pull-down of Lys80 RAD51 with biotinylated BRCA2Exon 27, BRCA2Exon 27-Ph and BRC4 peptides, visualized by anti-RAD51 western blot.
Figure 5
Figure 5
BRCA2Exon 27 binds to RAD51 nucleoprotein filaments. (a) Electrophoretic mobility shift assay testing the effect of BRCA2Exon 27 upon RAD51 nucleoprotein filaments formed on (b) φX174 virion ssDNA, (c) poly(dT) ssDNA, and (d) ds-ssDNA. In the case of the ds-ssDNA substrate, the I287T RAD51 protein was used. In each case, the RAD51 nucleoprotein filaments were also incubated separately with BRC4, as a control. (e) Electrophoretic mobility of fluorescein-labeled Fl-BRCA2Exon 27 upon the addition of RAD51, preformed nucleoprotein filaments between RAD51 and poly(dT) ssDNA, and poly(dT) ssDNA.
Figure 6
Figure 6
BRCA2Exon 27 protects RAD51 nucleoprotein filaments. (a) Electrophoretic mobility shift assay testing the ability of BRCA2Exon 27 and BRCA2Exon 27-Ph to protect RAD51 nucleoprotein filaments from disruption by BRC4. (b) Protection assay of RAD51 filaments formed on φX174 virion ssDNA using BRCA2Exon 27 and (c) BRCA2Exon 27-Ph. Lanes where the effect of BRCA2Exon 27 and BRCA2Exon 27-Ph is visible are highlighted by grey boxes. (d) Protection assay of RAD51 filaments formed on poly(dT) ssDNA using BRCA2Exon 27 and (e) BRCA2Exon 27-Ph. (f) Protection assay of filaments formed between I287T RAD51 and ds-ssDNA using BRCA2Exon 27 and (g) BRCA2Exon 27-Ph.
Figure 7
Figure 7
Electron microscopy of RAD51 nucleoprotein filaments. (a) RAD51–ssDNA filaments formed between RAD51 and poly(dT). (b) Incubation of pre-formed RAD51–poly(dT) filaments with BRCA2Exon 27. (c) Incubation of pre-formed RAD51–poly(dT) filaments with BRC4. (d) Incubation of RAD51–poly(dT) complexes with BRCA2Exon 27 prior to the addition of BRC4.
Figure 8
Figure 8
BRCA2Exon 27 protection is specific for ssDNA and independent of ATP hydrolysis. Electrophoretic mobility shift assay testing the ability of BRCA2Exon 27 to protect (a) the RAD51 oligomer in the absence of DNA, and nucleoprotein filaments formed between RAD51 and (b) poly(dT) ssDNA in the presence of AMP-PNP, (c) φX174 dsDNA in the presence of ATP, (d) 1.2kbp linear dsDNA in the presence of ATP and (e) 1.2kbp linear dsDNA in the presence of AMP-PNP. The dependence on nucleotide hydrolysis of the BRCA2Exon 27 protection effect can be assessed by comparing panel b with Figure 6d.
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References

    1. Rahman N, Stratton M. The genetics of breast cancer susceptibility. Annu Rev Genet. 1998;32:95–121. - PubMed
    1. Nathanson KL, Wooster R, Weber BL, Nathanson KN. Breast cancer genetics: what we know and what we need. Nat Med. 2001;7:552–6. - PubMed
    1. Connor F, et al. Tumorigenesis and a DNA repair defect in mice with a truncating BRCA2 mutation. Nat. Genet. 1997;17:423–430. - PubMed
    1. Patel KJ, et al. Involvement of BRCA2 in DNA repair. Mol Cell. 1998:347–357. - PubMed
    1. Moynahan ME, Pierce AJ, Jasin M. BRCA2 is required for homology-directed repair of chromosomal breaks. Mol Cell. 2001;2:511–518. - PubMed

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